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Method for the visual detection of specific antibodies in human serum by
the use of lateral flow assays

Abstract

This invention discloses a method and composition for detecting the
presence of class specific antibodies reactive with analytes such as
bacteria, allergens, autoimmune antigens, viral proteins, and
carbohydrates by lateral flow techniques. In one embodiment of the
invention, a test sample obtained from bodily fluids reacts with a gold
labeled antigen. The resulting complex travels across the membrane, and
along the lateral flow strip. Red colored lines formed in specific
locations along the test strip indicate the presence of class specific
antibodies in the test specimen. In another embodiment of the invention,
the lateral flow assay serves as an immunochromatographic screening test
for the detection of allergen-specific IgE antibodies in human serum.
Test sample reacts with gold labeled anti-IgE antibody. The resulting
complex travels across the membrane where immobilized allergens capture
the allergen specific IgE complex. Colored lines are formed int the test
areas to indicate the presence of allergen-specific IgE antibodies.

1) A lateral flow immunoassay device for detecting immunoreactions, said
device comprising: a test strip, said test strip comprising: a sample
site for applying a sample comprising biologically active materials, said
biologically active materials selected from the group consisting of
antigens and antibodies; a colorimetric labeling site for labeling the
sample, forming a calorimetric immuno complex, said colorimetric labeling
site comprising a colorimetric label positioned downstream from said
sample site; and at least one reaction site positioned downstream from
said calorimetric labeling site, said at least one reaction site
comprising reactive biological substances which binds said colorimetric
immuno-complexes when said biologically active materials of said sample
react positively with the biological substances.

2) The lateral flow immunoassay device of claim 1, wherein said test strip
further comprises a membrane support having a testing layer on the
surface thereof, wherein said membrane support is comprised of a material
selected from the group consisting of plastic, cardboard, nitrocellulose
and combinations thereof.

3) The lateral flow immunoassay device of claim 2, wherein said testing
layer further comprises a sample site to which the sample is applied.

4) The lateral flow immunoassay device of claim 3, further comprising a
sample pad residing on top of the testing layer, to which the sample is
transferred.

5) The lateral flow immunoassay device of claim 1, wherein said
calorimetric labeling site is a calorimetric labeled analyte to which the
sample is being tested.

6) The lateral flow immunoassay device of claim 5, wherein said
colorimetric label is selected from the group consisting of a metal sol
particle, a dyed labeled microparticle, a fluorescent labeled
microparticle, and combinations thereof.

7) The lateral flow immunoassay device of claim 6, wherein said metal sol
particle may be selected from the group consisting of platinum, gold,
silver, selenium, and copper.

10) The lateral flow immunoassay device of claim 1, further comprising at
least one binding site downstream from said calorimetric or fluorometric
labeling site.

11) The lateral flow immunoassay device of claim 10, wherein said at least
one reaction site comprises at least three reaction sites, said reaction
sites comprising: an anti-IgA binding site; an anti-IgM binding site; and
an anti-IgG binding site.

12) The lateral flow immunoassay device of claim 11, further comprising a
control site, said control site containing substances that readily react
with any calorimetric or fluorometric compound.

14) The lateral flow immunoassay device of claim 13, wherein said anti-Ig
immunoglobulin antibodies are from affinity purification of immune sera
selected from the group consisting of goats, rabbits, donkeys, sheep,
chickens, and other animals.

22) The lateral flow immunoassay device of claim 21, further comprising a
plurality of said reaction sites, each said reaction site being an
allergen site, wherein each allergen site has a different immobilized
allergen.

25) The lateral flow immunoassay device of claim 22, wherein said
allergens are immobilized on the test strip by the use of solubilizing
agents.

26) The lateral flow immunoassay device of claim 25, wherein said
solubilizing agents are selected from the group consisting of sugars and
alcohols, which allow for the unfolding of the allergen protein tertiary
structure such that more hydrophobic domains are exposed allowing
increased binding to the membrane, and increased reactivity to the IgE
antibody.

27) A lateral flow immunoassay device for detecting immune reactants, said
device comprising: a test strip, said test strip comprising: a sample
site for applying a sample comprising antibodies; a calorimetric labeling
site for labeling the sample, forming a calorimetric antibody complex,
said calorimetric labeling site comprising a calorimetric labeled analyte
positioned downstream from said sample site ; and at least four reaction
sites positioned downstream from said calorimetric labeling site, wherein
said there is a reaction site for detecting IgG antibodies, a reaction
site for detecting IgA antibodies, a reaction site for detecting IgM
antibodies, and a control reaction site, wherein each said reaction site
binds said colorimetric immune-antibody complexes when said antibodies of
said sample specific for each said reaction site are present in said
sample, whereupon a colored line will appear at each reaction site where
said antibodies of said sample have been bound to said colorimetric
labeled analyte.

28) The lateral flow immunoassay device of claim 27, wherein said
calorimetric label is selected from the group consisting of a metal sol
particle, a dyed labeled microparticle, a fluorescent labeled
microparticle, and combinations thereof.

29) The lateral flow immunoassay device of claim 28, wherein said metal
sol particle may be selected from the group consisting of platinum, gold,
silver, selenium, and copper.

34) The lateral flow immunoassay device of claim 33, wherein said anti-Ig
immunoglobulin antibodies are from affinity purification of immune sera
selected from the group consisting of goats, rabbits, donkeys, sheep,
chickens, and other animals.

41) A lateral flow immunoassay device for detecting immune reactants, said
device comprising: a test strip, said test strip comprising: a sample
site for applying a sample comprising antibodies; a colorimetric labeling
site for labeling the sample, forming a calorimetric antibody complex,
said colorimetric labeling site comprising a calorimetric labeled
anti-IgE antibody, said colorimetric labeling site positioned downstream
from said sample site ; and a plurality of reaction sites downstream from
said labeling site, each said reaction site containing a different
allergen such that when IgE antibodies labeled with colorimetric labeled
anti-IgE antibodies come in contact with an antigen to which the IgE
antibodies react, the reaction site will develop a colored line,
indicating a positive response.

43) The lateral flow immunoassay device of claim 41, further comprising a
plurality of said reaction sites, each said reaction site being an
allergen site, wherein each allergen site has a different immobilized
allergen.

46) The lateral flow immunoassay device of claim 43, wherein said
allergens are immobilized on the test strip by the use of solubilizing
agents.

47) The lateral flow immunoassay device of claim 46, wherein said
solubilizing agents are selected from the group consisting of sugars and
alcohols, which allow for the unfolding of the allergen protein tertiary
structure such that more hydrophobic domains are exposed allowing
increased binding to the membrane, and increased reactivity to the IgE
antibody.

48) A lateral flow immunoassay device for detecting immune reactants, said
device comprising: a test strip, said test strip comprising: a sample
site for applying a sample comprising antibodies; a fluorescent labeling
site for labeling the sample, forming a fluorescent antibody complexes,
said fluorometric labeling site comprising a fluorescent labeled analyte
positioned downstream from said sample site; and at least four reaction
sites positioned downstream from said fluorescent labeling site, wherein
said there is a reaction site for detecting IgG antibodies, a reaction
site for detecting IgA antibodies, a reaction site for detecting IgM
antibodies, and a control reaction site, wherein each said reaction site
binds said fluorescent antibody complexes when said antibodies of said
sample specific for each said reaction site are present in said sample,
whereupon a fluorescent line appears at each reaction site where said
antibodies of said sample have been bound to said floursescent labeled
analyte.

53) The lateral flow immunoassay device of claim 52, wherein said anti-Ig
immunoglobulin antibodies are from affinity purification of immune sera
selected from the group consisting of goats, rabbits, donkeys, sheep,
chickens, and other animals.

60) A lateral flow immunoassay device for detecting immunoreactants, said
device comprising: a test strip, said test strip comprising: a sample
site for applying a sample comprising antibodies; a fluorescent labeling
site for labeling the sample, forming a fluorescent antibody complex,
said fluorescent labeling site comprising a flouroscent labeled anti-IgE
antibody, said fluorescent labeling site positioned downstream from said
sample site ; and a plurality of reaction sites downstream from said
labeling site, each said reaction site containing a different allergen
such that when IgE antibodies labeled with fluorescent labeled anti-IgE
antibodies come in contact with an antigen to which the IgE antibodies
react, the reaction site will develop a flourescent line, indicating a
positive response.

62) The lateral flow immunoassay device of claim 60, further comprising a
plurality of said reaction sites, each said reaction site being an
allergen site, wherein each allergen site has a different immobilized
allergen.

65) The lateral flow immunoassay device of claim 62, wherein said
allergens are immobilized on the test strip by the use of solubilizing
agents.

66) The lateral flow immunoassay device of claim 65, wherein said
solubilizing agents are selected from the group consisting of sugars and
alcohols, which allow for the unfolding of the allergen protein tertiary
structure such that more hydrophobic domains are exposed allowing
increased binding to the membrane, and increased reactivity to the IgE
antibody.

Description

DESCRIPTION

[0001] This following application is a divisional of U.S. application Ser.
No. 09/689,682, filed Oct. 13, 2000.

[0009] d) A pad of absorbent bibulous material (the absorbent pad)
enclosed at the end opposite the sample well and used to absorb
transversely flowing sample, buffers and colloids;

[0010] e) A strip of bibulous material used in the sample well end to
initially absorb the sample being applied;

[0011] f) A strip of bibulous material in contact with the sample well
material and the lateral flow strip and containing a dried colored solid
phase reagent, the solid phase coated with proteins or haptens.

[0013] In the other chromatographic immunoassay, the analyte being
detected is human antibody specifically reactive with agents such as
viral/bacterial proteins (HIV, Hepatitis A and C, H. pylori, EBV,
Rubella, CMV, HSV, Dengue fever, Lyme, Chagas, TB, Toxoplasma, autoimmune
antigens, etc.) or allergens (pollens, molds, dust/mites, foods, animal
epithelia, etc.). The various analytes are abbreviated VB for simplified
use below. When it comes to detecting antibody, three formats are
typically used:

[0014] 1) The colored solid phase [SP] is coated with proteins or lectins
[protein A, protein G, lentil lectin, jacalin, concanavilin A, mannan
binding protein, wheat germ lectin, peanut lectin and avidchrom] that
react with human IgG antibodies. The solid phase may be coated with
anti-immunoglobulins that specifically react with IgG, IgM, IgA, or IgE.
The bibulous strip would in this case contain the analyte (VB) of
interest to which the specific antibody contained in the sample reacts.

[0015] 2) The colored solid phase contains the analyte (VB) to which the
human immunoglobulins react. The bibulous strip would in this case also
contain the analyte (VB) of interest to which the specific antibody
contained in the sample reacts.

[0017] U.S. Pat. No. 5,459,041 (Blaser et al.) discloses antigenic
compositions for use in diagnostic kits and the like for detecting the
presence of antibodies specific for Campylobacter pylori, Samples of
bodily fluids, for instance, may be contacted with immobilized antigen on
a solid phase which is then washed and tested for the occurrence of
significant levels of antigen/antibody complex. Levels exceeding a
predetermined positive threshold are indicative of antibodies to
Campylobacter pylori in the sample tested. Kits employing the antigenic
compositions of the invention preferably include means for detecting the
antigen/antibody complex such as materials and reagents for conducting an
enzyme-linked immunosorbent assay, Western blot technique, ELISA,
liposome-based assay or other known detection tests. The Western blot and
ELISA tests used here are for the detection of IgA and IgG antibodies.

[0018] U.S. Pat. No. 5,567,594 (Calenoff) discloses a library of isolated
and purified antigens specific for a microorganism is a set of individual
molecules. The library forms antigen-antibody complexes useful in the
context of diagnosing and treating conditions associated with a specific
microorganism such as H. pylori-induced gastro-duodenal disease. For the
antigen-antibody complexes the antibody in question is an immunoglobulin,
which is IgE if the antigens are allergens. Antigen-antibody complexes
with IgA, IgG and IgM are also useful if the antigen is a bacteria.. By
this multivariate approach, a specific condition is diagnosed with high
sensitivity and specificity by determining whether complexes form between
a specific antigen library and a biological sample which contains
immunoglobulins from an individual. Such libraries also are useful for
immunotherapy. Western blot is used to detect IgE antibodies. The method
requires enzyme conjugates and enzyme substrates and two wash steps to
detect antibodies.

[0019] U.S. Pat. No. 5,420,014 (Cripps et al.) discloses a method for
detecting a current infection by H. pylori in a mammal. The method
comprises contacting a mucous secretion [saliva] from said mammal with an
immobilized antigen component from H. pylori for a time and under
conditions sufficient for an IgG antibody in said mucous secretion
specific to a antigen component to form a complex therewith and then
subjecting said complex to a detecting means which involves an enzyme
conjugate and specific substrate.

[0020] U.S. Pat. No. 6,068,985 (Cripps) discloses a method which uses
saliva to detect IgG in both the Western Blot and ELISA tests. This
detection method requires the use of an enzyme conjugate and enzyme
substrate and two wash steps to detect the antibody.

[0021] U.S. Pat. No. 5,846,751 (Pronovost et al.) discloses a sensitive
and specific antigen preparation for the detection of Helicobacter pylori
in biological samples. The preparation uses a range of antigens derived
from size exclusion chromatography of detergent-solubilized H. pylori
cells and the purified antigen preparation is coated on the solid phase.
Serological assays such as ELISA, latex agglutination, and rapid EIA
assays are used to detect antibodies to H. pylori. The invention also
uses a lateral flow device to detect total immunoglobulins to H. pylori.
In this case, the H. Pylori antigen is striped on the membrane reaction
area and also coated to the colored solid phase. The antibody in the
sample reacts first with H. pylori gold coated conjugate, and then
travels to the membrane reaction area where it reacts with striped H.
pylori.

[0022] U.S. Pat. No. 5,200,344 (Blaser et al) uses a purified p28kd
protein from H. pylori to detect IgA, IgM and IgG antibody in ELISA and
Western Blot. The test requires conjugate and enzyme substrate and two
wash steps to detect the antibody.

[0025] None of these patents teach or disclose a fast and effective
lateral flow assay test for the testing of multiple-class specific
antibodies. More specifically, no chromatographic immunoassay is able to
distinguish between reactive antibody contained in the classes of human
antibody (IgG, IgA, IgM, IgD and IgE). All devices to date detect either
total immunoglobulins or IgG. The problem of separating reactivities of
antibody class lies in the 10 to 15 fold excess of IgG class specific
antibodies over IgA, IgM, and IgE class specific antibodies reactive with
analyte (VB) in question at various protein sites (epitopes). If the IgG
is allowed to react at the same time or same rate as other classes of
antibody, the IgG will mask most if not all the analyte (VB) epitopes,
thereby decreasing or eliminating the activity of the IgM, IgA, and IgE
class antibodies to the analyte (VB).

SUMMARY OF THE INVENTION

[0026] The proposed invention allows for antibody class recognition. In
one embodiment of the invention, a lateral flow immunoassay device
distinguishes at least three classes of antibody. The classes of antibody
to be distinguished include IgG, IgA and IgM. A control line reactive
with gold particles is also present. .

[0027] In another embodiment of the invention, the immunoassay test strip
is modified to allow detection of the IgE class of antibody to many
allergens (VB) coated sequentially on a bibulous strip. Saturated
anti-IgE antibodies coated to colored solid [SP] phase particles at high
concentration are reacted with a controlled amount of serum to allow for
the near complete complexing of elevated levels of human or other animal
IgE. This insures that little free IgE is left unreacted. Unreacted IgE
would inhibit the reaction with the multiple analytes coated on the
bibulous strip. By capturing most of the IgE on the colored solid phase,
sufficient IgE specific antibody molecules are available to react with
the various allergens (VB) as the reaction front moves transversely down
the strip toward the absorbent pad. This allows for the detection of many
different IgE allergen specific molecules.

[0028] In both embodiments of the inventions, an IgG reacting protein
(which can be protein A, protein G, an antibody to IgG or lectins such as
lentil lectin, jacalin, concanavilin A, mannan binding protein, wheat
germ lectin, peanut lectin and avidchrom) is added to the sample pad in
order to complex the IgG contained in the sample such that the molecular
weight of the IgG complex is greater than 1.0 million. This large complex
travels sufficiently slower than IgA, IgM, and IgE thereby allowing these
antibodies to react prior to the IgG. After reacting to the antigen
coated colored solid phase, the various reacted complexes are captured on
the bibulous strip coated at three sites with antibody to IgM, IgA and
IgG or a protein/lectin reactive with IgG (protein A, protein G, lentil
lectin, jacalin, concanavilin A, mannan binding protein, wheat germ
lectin, peanut lectin and avidchrom). Thus, the class of reactive
antibody is distinguished.

[0029] In another embodiment of the invention, the colored solid phase
contains proteins that react with IgG, allowing for the detection of many
different analyte specific antibody molecules of the IgG class. The
reagents coated onto the bibulous lateral flow strip include autoimmune
antigens, allergens, Chlamidia, Rickettsiae, viruses, and bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The invention will be better understood and further objects,
characterizing features, details and advantages thereof will appear more
clearly as the following description proceeds with reference to the
accompanying diagrammatic drawings given by way of non limiting example
only illustrating a presently preferred specific embodiment of the
invention.

[0031] FIG. 1 is a perspective view of one embodiment of the lateral flow
immunoassay test;

[0032] FIG. 2 is a perspective view of another embodiment of the lateral
flow immunoassay test;

[0033] FIG. 3 is an exploded view of the lateral flow immunoassay test;

[0034] FIG. 4 is a side view of the test strip;

[0035] FIG. 5 is a view of the colored particles attached to the antigen;
and

[0036] FIG. 6 is an exploded view of another version of the lateral flow
immunoassay test.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The immunoassay lateral flow test system 1 comprises a casing 2,
preferably plastic, surrounding a test strip 7. On the top surface of the
test strip there is a sample opening 8 and a test results opening 9 to
show the results of the assay. The casing can take a number of different
forms. In FIGS. 3 and 6,, the casing has a top section 4 or 100, and a
bottom section 5 or 101. Within the top section 4 there is at least one
channel 6 into which is fitted a test strip 7. The test strip 7
preferably has a membrane support 10. The membrane support 10 may be
comprised of plastic, cardboard, or any other rigid material. On top of
the membrane support 10 is a testing layer 11, preferably made out of
nitrocellulose. On top of the nitrocellulose or testing layer 11 are the
areas to which the appropriate reagents or samples are applied or
affixed. The nitrocellulose/testing layer may be affixed to the membrane
support 10.

[0038] At one end of the test strip 7 is the sample site 12 to which the
sample is to be applied. This sample site 12 preferably has a sample pad
13 residing on top of the testing layer, to which the sample is
transferred. The sample is preferably a bodily fluid. This fluid may be
serum whole blood, plasma, colostrum, milk, saliva, tears, or urine
sample from a human or other animal species.

[0039] Incorporated in the sample site 12 or sample pad 13, or downstream
from the sample site is the labeled antigen, for which the serum is being
tested.

[0040] The gold particles 14 attached to the antigen or antibody 15 are
preferably larger than 20 nm, more preferably in the range of about 20 to
100 nm, and most preferably in the range of 20 to 40 nm. The gold sol
labeled antigens/antibodies 35 are dried and deposited on the strip 7.

[0041] The metal sol particles to be used in accordance with the present
invention may be prepared by coupling the analyte directly to the gold
particle. Additionally, the labeled component may be prepared by coupling
the analyte to the particle using a biotin/avidin linkage. In this latter
regard, the substance may be biotinylated and the metal containing
particle coated with an avidin compound. The biotin on the analyte may
then be reacted with the avidin compound on the particle to couple the
substance and the particle together. In another alternative form of the
invention, the labeled component may be prepared by coupling the analyte
to a carrier such as bovine serum albumin (BSA),key hole lymphocyananin
(KLH), or ovalbumin and using this to bind to the metal particles.

[0042] The metal sol particles to be used in accordance with the present
invention may be prepared by methodologies which are well known. For
instance, the preparation of gold sol particles is disclosed in an
article by G. Frens, Nature, 241, 20-22 (1973). Additionally, the metal
sol particles may be metal or metal compounds or polymer nuclei coated
with metals or metal compounds, as described in U.S. Pat No. 4,313,734.
Other methods well known in the art may be used to attach the analyte to
gold particles. The methods include but are not limited to covalent
coupling and hydrophobic bonding. The metal sol particles may be made of
platinum, gold, silver, selenium, or copper or any number of metal
compounds which exhibit characteristic colors.

[0043] Similarly, the analyte does not necessarily have to be attached to
a metal sol particle, but may instead be attached to dyed or fluorescent
labeled microparticles such as latex, polystyrene, dextran, silica,
polycarbonate, methylmethacrylates and carbon.. The metal sol particles,
dyed or fluorescent labeled microparticles should be visible to the naked
eye or able to be read with an appropriate instrument (spectrophotometer,
fluorescent reader, etc. ).

[0044] There are a number of ways in which the gold labeled antigens 16
may be deposited on the strip 7.

[0045] In an alternative and preferred embodiment, the gold labeled
antigens/antibodies are deposited and dried on a rectangular or square
absorbent pad 16, the pad preferably about 0.25".times.0.25"or less. This
absorbent pad 16 is positioned downstream from where the sample is
applied on the strip 7.

[0046] In yet another embodiment of the invention, the analytes may be
attached to microspheres. This has the effect of increasing the number of
reactive sites (epitopes) in a given area. Analytes may be attached to
these alternate solid phases by various methodologies.

[0047] For instance, reactive microspheres (MX-Covaspheres.RTM. of
diameter 0.5 micrometers or 0.9 micrometers) purchased from Duke
Scientific Corporation, Pal Alto, Calif. 94303, or other suppliers, may
be used to covalently attach analytes. The binding is at the amino groups
of the protein if covalent methodology is used. In addition, hydrophobic
or electrostatic domains in the protein may be used for passive coating.
A suspension of the spheres is mixed after sonication with the
antigens/antibodies in water or in a phosphate buffer solution, after
which they are incubated at room temperature for 10-75 minutes. The
mixture is then centrifuged and the pellets containing the
antigen/antibody-linked microspheres are suspended in a buffer containing
1-5% wt/volume bovine serum albumin (BSA) for 1 hour at room temperature.
The BSA blocks any unreacted surfaces of the microspheres. After one more
centrifugation, the spheres are resuspended in buffer (TBS with 5% BSA)
and stored at 4 degrees C. before using.

[0048] The solid phase particles may comprise any one of known, water
dispersable particles, such as, the polystyrene latex particles disclosed
in U.S. Pat. No. 3,088,875. Such solid phase materials simply consist of
suspensions of small, water-insoluble particles to which
antigens/antibodies are able to bind. Suitable solid phase particles are
also disclosed, for example, in U.S. Pat. Nos. 4,184,849; 4,486,530; and
4,636,479.

[0049] In another embodiment of the invention, the analytes may be
attached to fluorescent microspheres or fluorescent microparticles. Said
fluorescent micropaticles may be purchased from Duke Scientific, Palo
Alta, Calif. 94303 and are listed as Green, Red, or Blue fluorescent 0.4
micron microspheres (Product Bulletin 93). They are also available from
Molecular Probes, Eugene, Oreg. 97402 and are listed as FluoroSpheres;
Blue, Yellow-Green, Nile Red, Orange, Red, Crimson, Dark Red and Far Red
in micron sizes from 0.03 to 5.0. Other manufactures also supply
fluorescent microspheres. Characteristically, fluorescent microspheres
incorporate fluorescent dyes in the solid outer matrix or in the internal
volume of the microsphere. The fluorescent spheres are typically detected
by a fluorescent reader that excites molecules at one wavelength and
detects the emission of fluorescent waves at another wavelength. For
example, Molecular Probes Nile Red particles excite at 526 nm at emit at
574 nm, the Far Red excites at 680 nm and emits at 720 nm and the Blue
excites at 365 nm and emits at 430 nm. In a lateral flow format,
detection of fluorescent microparticles requires the use of a reflectance
reader with an appropriate excitation source (HeNe, Argon, tungsten or
diode laser) and an appropriate emission filter for detection. Use of
diode lasers allows for use of detection systems that use low cost lasers
with detection above 600 nm. Most background fluorescence is from
molecules that emit fluorescence below 550 nm.

[0050] Fluorescent microspheres contain surface functional groups such as
carboxylate, sulfate and aldehyde groups, making them suitable for
covalent coupling of proteins and other amine containing biomolecules. In
addition, sulfate, carboxyl and amidine microspheres are hydrophobic
particles that will passively absorb almost any protein or lectin.
Coating is thus similar as for non fluorescent microspheres
(Mx-Covaspheres or other latex microparticles). A suspension of the
fluorescent spheres is mixed after sonication with the antigens/antibody
in water or in a phosphate buffered solution, after which they are
incubated at room temperature for 10-75 minutes. EDAC (soluble
carbodiimide), succinimidyl esters and isothiocyanates as well as other
crosslinking agents may be used for covalent coupling of proteins and
lectins to the microspheres. After the protein has attached to the
surface of the miroparticles, the mixture is centrifuged and the pellets
containing the antigen or antibody linked to the fluorescent
microparticles are suspended in a buffer containing 1-5% bovine serum
albumin for one hour. After one more centrifugation, the spheres are
resuspended in buffer (TBS with 5% BSA or other appropriate buffers) and
stored at 4 degrees C. before use.

[0051] The solid phase particles useful in connection with the invention
may comprise, for example, particles of latex or of other support
materials such as silica, agarose, glass, polyacrylamides, polymethyl
methacrylates, carboxylate modified latex and Sepharose. Preferably, the
particles will vary in size from about 0.2 microns to about 10 microns.
In particular, useful commercially available materials include 0.99
micron carboxylate modified latex, cyanogen bromide activated Sepharose
beads (Sigma), fused silica particles (Ciba Corning, lot #6),
isothiocyanate glass (Sigma), Reactogel 25DF (Pierce) and
Polybead--carboxylate monodisperse microspheres. In accordance with the
invention, such particles may be coated with a layer of antigens coupled
thereto in a manner known per se in the art to present the solid phase
component.

[0052] In the preferred embodiment, the sample contains antibodies which
will react with the gold labeled antigen, forming an antigen-antibody
complex. The gold-antigen antibody complex begins to migrate along the
test strip.

[0053] Further down the length of the test strip are four binding sites.
The first binding site 18 is preferably to bind IgM. The second binding
site 19 is preferably a site to bind IgA. The third binding site 20 is
for the binding of IgG and the fourth binding site 22 is for a control.
More specifically, each binding site is in the form of a striped line
along the width of the test strip opening 9. At the site of each binding
site, there are anti-Ig immunoglobulins. For example, class specific
antibodies are laid down on the test strip. For example, a goat
anti-human IgM antibody is laid down at the first binding site 18, goat
anti-human IgA antibody is laid down at the second site 19 and goat
anti-human IgG antibody is laid down at the third binding site 20. At the
control site there is immobilized a protein or substance containing
sulfur residues that readily react with any colloidal gold compound. It
can also be an antibody reactive with the proteins coated on the gold or
microparticles surface. Since the gold or microparticles conjugate is
always in excess of sample reactive antibodies, sufficient conjugate is
available to react with the control line. The antibodies reactive with
IgM, IgA and IgG can be from affinity purification of immune sera from
goats, rabbits, donkeys, sheep, chickens or other animals. It may also be
monoclonal antibodies directed against IgM, IgA and IgG. The antibodies
used are specific for the heavy chain portion of the IgM, IgA and IgG
antibodies. Substances reactive with IgG (protein A, protein G, lentil
lectin, jacalin, concanavilin A, mannan binding protein, wheat germ
lectin, peanut lectin and avidchrom) may be substituted for the antibody
to IgG or combined with said antibody.

[0054] After the sample has been placed at the sample opening 8, the
sample migrates to the site of the gold sol labeled analytes pad 16.
Analytes specific for the gold sol conjugate will attach and bind, thus
forming gold sol labeled complexes. The gold sol complex continues to
migrate along the length of the lateral flow strip. Reactive complexes
are specifically captured by analyte (VB) coated on the test strip 7.
Migration continues and complexes are captured on the control line of the
test strip 7. Excess fluid is wicked into the absorbent pad 17.

[0055] The problem of separating reactivities of antibody classes lies in
the 10 to 15 fold excess of IgG over IgA and IgM specific antibody
reactivity with analyte reaction sites. If the IgG is allowed to react at
the same time or rate as other classes of antibody, the IgG will mask
most if not all the analyte epitopes, thereby decreasing or eliminating
the activity of the IgM and IgA class antibodies to the analyte.

[0056] To solve this problem, an IgG reacting substance ( which can be,
among others, protein A, protein G, an antibody to IgG, lentil lectin,
jacalin, concanavilin A, mannan binding protein, wheat germ lectin,
peanut lectin and avidchrom) is added to the sample pad in order to
complex the IgG such that the molecular weight of the complex is greater
than 1 million. This large complex travels sufficiently slower than IgA,
IgM, and IgE, thereby allowing these antibodies to react prior to the
IgG. After reacting with the colored solid phase, the various reacted
complexes are captured specifically at three sites by the antibodies to
IgM, IgA,, and IgG, or a substance reactive with IgG (protein A, protein
G, lentil lectin, jacalin, concanavilin A, mannan binding protein, wheat
germ lectin, peanut lectin and avidchrom).

[0057] For example, to determine whether a person has been exposed to
Helicobacter pylori, or to determine if there has been successful
treatment of the disease, a serum sample is tested to determine whether
it contains antibodies to H.pylori. Assuming the use of gold labels, if
lines appear at the binding sites for IgG and weakly for IgM, then there
is only a chronic condition present. If however, lines appear at the
binding sites for IgM and IgA, with or without IgG, then an active or
recent colonizing infection is occurring. The detection of IgA when
combined with a low serum pepsinogen level is associated with an
increased risk of gastric cancer.

[0059] In another embodiment of the invention, this lateral flow assay can
be used for the visual detection of allergen specific IgE antibodies in
human or animal serum. In this assay the test serum reacts with a
colorimetric (preferably gold) labeled anti-IgE antibody contained in the
colorimetric (preferably dried) gold pad. 16. The resulting complex
travels along the test strip to the line stripped allergen site 24. At
the allergen site, there are a plurality of immobilized allergens 24.
Indeed, the immunoassay can easily test for one or more different
allergens, preferably by one strip 25, two strips (25 and 26) or multiple
strips next to each other. Each strip can contain one or more specific
allergen lines. The common allergens which may be tested include but are
not limited to pollens (Timothy, cultivated rye, birch, alder, hazelnut,
mugwort, English plantain, ragweed, nettle, etc.), dust allergens (D.
farinae, D. pteronyssinus, house dust), molds (Alternaria tenuis,
Aspergillus fum., Cladosporium, Penicillium not), animal epithelium (Cat
epithelium, dog dander, horse dander, goose feathers) foods (dairy,
cereals, nuts, seafoods, legumes and mixes thereof), inhalant mixes
(pollen I (grasses), pollen II (weed/trees), animal mix, dust mix, mold
mix) and combinations thereof. The allergens are immobilized on the test
strip by the use of solubilizing agents such as sugars and alcohols
(sucrose, mannose, fructose, ethylene glycol, ethanol, methanol,
glycerin, dextrans). The use of sugars and alcohols unfolds the allergen
protein tertiary structure such that more hydrophobic domains are exposed
allowing greater binding to the membrane. In addition, protein to protein
aggregation is reduced through solubilization allowing individual protein
molecules to bind to the nitrocellulose or nylon membrane.

[0060] Assuming there is a reaction between the complexes of gold labeled
anti IgE antibody and the sample containing IgE antibody and the
allergens, a red line will appear at the site of the allergen when there
is a positive response. The assay validity is demonstrated by the
appearance of a red colored line in the positive control region of the
membrane. The positive control is a protein or substance containing
sulfur residues that readily react with any colloidal gold compound. It
can also be an antibody reactive with the proteins coated on the gold or
microparticles surface. Since the gold or microparticles conjugate is in
excess, sufficient conjugate is available to react with the control line.

[0061] Many modifications and variations of the present invention are
possible in light of the above teachings. It is, therefore, to be
understood within the scope of the appended claims the invention may be
protected otherwise than as specifically described.